Bisphenol a and Bisphenol S Disruptions of the Mouse Placenta and Potential Effects on the Placenta–Brain Axis

Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta–brain axis

Jiude Maoa,b, Ashish Jainc,d, Nancy D. Denslowe,f, Mohammad-Zaman Nourie,f, Sixue Cheng,h, Tingting Wangh, Ning Zhuh, Jin Kohh, Saurav J. Sarmaa,i, Barbara W. Sumnera,i, Zhentian Leia,i,j, Lloyd W. Sumnera,i,j, Nathan J. Bivensk, R. Michael Robertsa,j,l,1, Geetu Tutejac,d,1, and Cheryl S. Rosenfelda,b,m,n,1

aBond Life Sciences Center, University of Missouri, Columbia, MO 65211; bBiomedical Sciences, University of Missouri, Columbia, MO 65211; cBioinformatics and Computational Biology, Iowa State University, Ames, IA 50011; dGenetics, Development and Cell Biology, Iowa State University, Ames, IA 50011; ePhysiological Sciences, University of Florida, Gainesville, FL 32611; fCenter for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611; gDepartment of Biology, Genetics Institute, University of Florida, Gainesville, FL 32610; hProteomics and Mass Spectrometry Facility, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610; iUniversity of Missouri Metabolomics Center, University of Missouri, Columbia, MO 65211; jBiochemistry, University of Missouri, Columbia, MO 65211; kDNA Core Facility, University of Missouri, Columbia, MO 65211; lAnimal Sciences, University of Missouri, Columbia, MO 65211; mThompson Center for Autism and Neurobehavioral Disorders, University of Missouri, Columbia, MO 65211; and nUniversity of Missouri Informatics Institute, University of Missouri, Columbia, MO 65211

Contributed by R. Michael Roberts, December 28, 2019 (sent for review November 7, 2019; reviewed by Alina Maloyan and Xiaoqin Ye) Placental trophoblast cells are potentially at risk from circulating chemical were manufactured (3). Approximately 93% of the US endocrine-disrupting chemicals, such as bisphenol A (BPA). To population has detectable amounts of BPA in their urine, suggestive understand how BPA and the reputedly more inert bisphenol S of widespread exposure (4). Exposure to BPA and bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 μg/kg (BPS) is primarily dietary (5, 6), although there are additional body weight BPA or BPS daily for 2 wk and then bred. They con- routes of contact (7, 8). Additionally, BPA can be readily trans- tinued to receive these chemicals until embryonic day 12.5, where- mitted from mother to offspring via the placenta (9, 10) and is, upon placental samples were collected and compared with therefore, a major environmental concern during pregnancy. Be- unexposed controls. BPA and BPS altered the expression of an iden- cause of consumer unease with BPA, more manufacturers are be- tical set of 13 genes. Both exposures led to a decrease in the area ginning to use BPA substitutes, such as BPS, as plasticizers in occupied by spongiotrophoblast relative to trophoblast giant cells household products. However, some reports indicate that (GCs) within the junctional zone, markedly reduced placental sero- such analogs induce similar outcomes as BPA (11, 12). tonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. In the recent decade, there have been several studies exam- Concentrations of dopamine and 5-hydroxyindoleacetic acid, the ining the effects of BPA on the mouse placenta (13–22). Other main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A + Significance strong positive correlation between 5-HT GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In con- The mammalian placenta is a target for endocrine-disrupting trast, a negative correlation existed between dopamine+ GCs and chemicals, such as the widely prevalent plasticizer bisphenol A reductions in spongiotrophoblast to GC area ratio. These outcomes (BPA). Consumer unease with BPA has led to manufacture of lead to the following conclusions. First, BPS exposure causes almost substitutes, such as bisphenol S (BPS). Here, we used a mul- identical placental effects as BPA. Second, a major target of BPA/BPS tiomics approach to study effects of developmental exposure is either spongiotrophoblast or GCs within the junctional zone. to BPA and BPS on midgestational mouse placenta. BPA and Third, imbalances in neurotransmitter-positive GCs and an observed BPS caused almost identical changes in gene expression; similar decrease in docosahexaenoic acid and estradiol, also occurring in morphological defects in the junctional zone, which forms the response to BPA/BPS exposure, likely affect the placental–brain axis interface of the placenta with maternal endometrium; and of the developing mouse fetus. marked alterations in placental content of neurotransmitters serotonin and dopamine. The results imply that in rodents there could be associated effects on the placental–fetal brain trophoblast | endocrine disruptor | transcriptomics | neurotransmitters | labyrinth axis. We also conclude that BPS should be regarded as haz- ardous as BPA.

he placenta is a transient organ that anchors the developing Author contributions: J.M., N.D.D., S.C., N.Z., L.W.S., R.M.R., G.T., and C.S.R. designed Tfetus to the wall of the reproductive tract and plays multi- research; J.M., M.-Z.N., T.W., N.Z., J.K., S.J.S., B.W.S., Z.L., and N.J.B. performed research; faceted roles in nutrient, gas, and waste exchange. In eutherian J.M., A.J., N.D.D., M.-Z.N., S.C., T.W., N.Z., J.K., S.J.S., B.W.S., Z.L., L.W.S., N.J.B., R.M.R., G.T., mammals, it produces a range of hormones and cytokine factors and C.S.R. analyzed data; and J.M., A.J., M.-Z.N., S.C., T.W., N.Z., J.K., S.J.S., B.W.S., Z.L., L.W.S., N.J.B., R.M.R., G.T., and C.S.R. wrote the paper. that couple the needs of the fetus to responses in the mother. Additionally, in many species, including rodents and humans, Reviewers: A.M., Oregon Health & Science University; and X.Y., University of Georgia. that have a hemochorial type of placentation, the syncytio- The authors declare no competing interest. trophoblast cells involved in nutrient and gas exchange become Published under the PNAS license. bathed in maternal blood (1), rendering them potentially vul- Data deposition: The RNA sequence data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database (accession no. 141322). The raw Gas Chro- nerable to toxicants circulating within the maternal bloodstream. matography Mass Spectrometry (GC/MS) metabolomics data are available at While the placenta detoxifies certain xenobiotic chemicals, as an Metabolomics Workbench (Project ID PR000891). endocrine organ, it can also be affected by those compounds that 1To whom correspondence may be addressed. Email: [email protected], geetu@ interfere with its endocrine functions. One such endocrine- iastate.edu, or [email protected]. disrupting chemical (EDC) that is widely prevalent in the envi- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ronment and present in various household items is bisphenol A doi:10.1073/pnas.1919563117/-/DCSupplemental. (BPA) (2). In 2013, it was estimated that 15 billion lbs. of this First published February 18, 2020.

4642–4652 | PNAS | March 3, 2020 | vol. 117 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1919563117 Downloaded by guest on September 25, 2021 studies have focused on the effects of BPA in isolated human Transcriptomic Analyses of Mouse Placental Tissues Exposed to BPA placenta lines and in term human placenta (23–33). A concern or BPS. RNA sequencing (RNAseq) analysis was performed on with using cells derived from human term tissues is that they fail placentas recovered from BPA- and BPS-treated and control to replicate the full range of placental responses associated with dams at embryonic day 12.5 (e12.5), and the raw data have been ∼ the critical early and midpregnancy periods (34). In addition, cell deposited (37). An average of 97.06% of reads ( 124 million) was lines derived from neoplasms (e.g., BeWo, JEG-3, and JAR) or mapped. The average mapped read count after filtering the reads transformed in some manner may not have physiological equiva- that mapped to random chromosomes and to mitochondrial DNA was ∼121 million (SI Appendix,TableS3). lence to their normal trophoblast counterparts. Previous mouse Principal component analyses (PCA) of the RNAseq data did studies have generally examined either transcriptomic responses – not show any clear separation based on either treatment or the or effects on placental morphology (13 22), but there are no de- interaction of treatment with sex (Fig. 1A). Thus, we combined finitive reports on the effects of the BPA analog, BPS (structures male and female placental results within the same group. Based SI Appendix are shown in ,Fig.S1). Additionally, sex differences on false discovery rate (FDR), only 11 genes were differentially have not been examined toward either chemical, even though the expressed (FDR ≤ 0.05; fold change ≥ 1.5; transcripts per mil- placenta is sexually dimorphic (1, 35). lion ≥ 1) relative to controls in the BPS-exposed placentas, and 3 Here, we have performed a comprehensive examination on genes were differentially expressed in the BPA-exposed pla- how maternal exposure to BPA and BPS influence mouse placentas (Table 1). On closer investigation of the differentially cental development by midpregnancy. In doing so, we have expressed genes (DEGs), both treatments influenced expression considered whether the two chemicals provoke closely similar or of a common set of 13 genes, of which 11 were down-regulated distinctly different placental responses and hence, whether one (P ≤ 0.05) and 2 (Actn2 and Efcab2) were modestly up-regulated chemical is potentially more benign than the other. Additionally, (P ≤ 0.05). The gene set was enriched for at least four genes with we examined the metabolomes of exposed and control placentas, transcripts that are typically augmented in placenta (Sfrp4, Coch, especially for the presence of neurotransmitters, because it is Gm9513,andCalm4) as determined by using the program now becoming evident that the placenta response to environ- TissueEnrich (38) (Fig. 1 B and C). One of them, Gm9513,has Pate11 mental insults likely influences development and function of a sequence similar to that encoded by the (prostate and other major fetal organs, especially the brain (1, 36). testis expressed 11) locus on chromosome 9 of the mouse (http://www.informatics.jax.org/marker/MGI:3779923)andisa PATE

Results paralog of human gene family members. In general, the APPLIED BIOLOGICAL SCIENCES General Maternal and Fetal Measures. Several outcomes in relation data shown in Table 1 suggest that BPA and BPS have almost to pregnancy were compared between the BPA/BPS treatment indistinguishable effects on gene expression in the e12.5 mouse groups and controls (SI Appendix, Tables S1 and S2). No dif- placenta but influence the expression of only a handful of ferences were observed between the BPA/BPS and control genes. There was no significant influence, as determined by FDR,ofsexonexpressionofthese13genesbasedonananalysis groups in pregnancy success, maternal gestational weight, num- of treatment × sex interactions (SI Appendix,TableS4). ber of implantation sites, and number of fetuses. Nor were there Protein–protein interactions involving RIMKIb to CALM4 differences in sex ratio when values were assessed according to and EPDR to SFRP4 were predicted by using the STRING deviation from the expected 1:1 ratio of males vs. females. Database (SI Appendix, Fig. S2). Functional enrichment analyses However, when conceptus sex ratios in BPA and BPS groups with the WEB-based Gene Set Analysis Toolkit (39) revealed were analyzed relative to each other and to controls, there was a that the Wingless Int-1 (Wnt) and chemokine signaling pathways, trend for BPA exposure to show increased percentage of male amino acid metabolism, and possibly, neurotransmission were likely conceptuses (56.9 ± 4.7 vs. 42.9 ± 7.9, P = 0.06). Additional affected by BPA/BPS exposures (SI Appendix,Fig.S3). experiments with many more animals would be needed to ex- qPCR, in general, confirmed the RNAseq data shown in Table plore further whether sex ratio is distorted by BPA treatment. 1. The one exception was Actn2, which appeared to be slightly

AB C

2.0 Srfp4

1.5 Log2(FPKM) Gm9513 5 BPA Pate-P 4 BPS and 3 (P-Adjusted) 1.0 Pate-11 2 CTL 10 Coch 1 0 -LOG 0.5

PC3 Calm4 −10 0 10 20 30 20 20

− 10 0.0 Liver Limb

0 Lung Heart Testis − Cortex Kidney −10 Spleen Thymus Intestine Placenta − Liver −20 Liver Limb PC2 Lung Brain Heart Heart Testis Cerebellum − E14.5−Liver E14.5 Cortex E14.5−Brain 40 −30 Kidney − Spleen −30 E14.5−Heart Bone Marrow − Thymus Olfactory bulb Olfactory −40 −20 0 20 40 60 80 Intestine E14.5 −Placenta Cerebellum E14.5− E14.5 E14.5− E14.5

PC1 Bone Marrow Olfactory bulb Olfactory E14.5

Fig. 1. Effects of BPA/BPS on placental gene expression. (A) PCA plot using the top quartile of the most variable genes across the samples. The color in the plot represents the different treatments, and sex is represented by the shape (females are triangles, and males are dots; each symbol represents a single biological replicate for a given group). TissueEnrich analyses of differentially expressed (DE) genes in BPA/BPA vs. control placenta. (B) Tissue-specific gene enrichment analysis of genes down-regulated in BPA or BPS relative to controls shows enrichment for genes with placenta-specific expression. (C) Heatmap showing expression levels for placenta-specific genes that were down-regulated in BPA or BPS relative to controls across tissues assayed in the mouse ENCODE Project. Placenta-specific genes were determined by TissueEnrich. Calm4, Coch, and Sfrp4 are highly expressed in placenta and a small subset of other tissues (group enriched), whereas Gm9513 is only expressed in the placenta. FPKM, fragments per kilobase of transcript per million mapped reads.

Mao et al. PNAS | March 3, 2020 | vol. 117 | no. 9 | 4643 Downloaded by guest on September 25, 2021 Table 1. DEGs in BPA or BPS vs. control placenta BPA BPS

Gene Log2 fold Fold Log2 fold Fold symbol Gene name P value FDR change change P value FDR change change

Actn2 Actinin α2 1.20E-02 6.46E-01 0.69 1.62 3.04E-05 4.45E-02 1.11 2.17 Calm4 Calmodulin 2 1.13E-03 3.17E-01 −3.26 −9.55 5.54E-06 1.78E-02 −4.49 −22.52 Coch Cochlin 5.02E-03 5.18E-01 −2.47 −5.55 1.27E-05 2.92E-02 −3.80 −13.91 Cxcl14 C-X-C Motif Chemokine 2.25E-06 1.81E-02 −3.85 −14.41 1.12E-04 1.05E-01 −3.10 −8.59 Ligand 14 Ear2/NR2F6 Nuclear Receptor Subfamily 2 5.41E-05 1.09E-01 −4.04 −16.46 5.47E-06 1.78E-02 −4.53 −23.08 Group F Member 6 Efcab2 EF-Hand Calcium Binding 5.49E-06 2.95E-02 0.59 1.51 8.20E-02 8.17E-01 0.23 1.17 Domain 2 Epdr1 Ependymin Related 1 1.36E-05 5.46E-02 −2.05 −4.14 2.33E-05 3.75E-02 −1.96 −3.91 Gdf10 Growth Differentiation 2.36E-05 7.60E-02 −2.36 −5.15 2.45E-06 1.32E-02 −2.61 −6.09 Factor 10 Gm9513/PATE1 Prostate and Testis 5.28E-03 1.00E+00 −2.16 −4.48 5.18E-06 2.37E-02 −3.54 −11.6 Expressed 1 Guca2a Guanylate cyclase activator 2a 7.21E-04 2.76E-01 −3.79 −13.84 2.05E-05 3.66E-02 −4.74 −26.72 (guanylin) Mmp3 Matrix Metallopeptidase 3 5.53E-04 2.62E-01 −2.67 −6.38 8.32E-06 2.23E-02 −3.46 −11.04 Rimklb Ribosomal Modification Protein RimK 1.42E-03 3.41E-01 −1.87 −3.67 1.50E-05 3.02E-02 −2.51 −5.7 Like Family Member B Sfrp4 Secreted Frizzled Related 3.67E-08 5.91E-04 −5.67 −51.05 1.83E-07 2.95E-03 −5.30 −39.49 Protein 4

up-regulated as determined by RNAseq but down-regulated by no effect of maternal treatment or maternal treatment × conceptus the criterion of qPCR. Based on a previous report that BPA sex interaction for another neurotransmitter, γ-aminobutyric acid exposure altered the expression of Ascl2 (Achaete-Scute Family (GABA) (SI Appendix,TableS6). Basic Helix–loop–helix Transcription Factor 2) (18) and the contributory role of this imprinted gene in differentiation of the Placental Estradiol, Estrone, Corticosterone, Testosterone, and mouse placenta layers (40), we also screened the expression of Progesterone Concentrations. Placental concentrations of estra- this gene by qPCR analysis. Both BPA and BPS exposure diol (E2) were modestly but significantly reduced in BPA-exposed appeared to reduce placental expression of Ascl2 modestly but placentas compared with the control group (P = 0.01) (Fig. 3D). with only BPA having a significant (P < 0.05) effect (Fig. 2). However, BPS exposure did not result in a significant effect. None RNAseq data indicated no differences for Ascl2 expression after of the other placental steroid hormones examined were affected exposure to either BPA or BPS. The fold change data for these by either maternal treatment or maternal treatment × conceptus genes based on treatment × sex interactions are shown in SI sex interactions (SI Appendix,TableS7). Appendix, Table S5. Again, no significant effect of fetal sex was observed. BPA/BPS-Induced Placental Histopathological Changes. Within each placental section, the areas of labyrinth, spongiotrophoblast zone, Nontargeted Metabolomics in Fetal Placental Tissue. A broad, non- and trophoblast giant cells (GCs) were measured. No interaction targeted analysis of polar metabolites extracted from e12.5 pla- of maternal treatment and conceptus sex was observed, and thus, centa was performed, and the raw data have been deposited (41). male and female placenta data within each group were combined These data revealed that BPA and BPS exposure resulted in a P ≤ to increase the power of the analyses. Exposure to either BPA or number of apparent changes based on values 0.05 (Dataset BPS reduced the ratio of the spongiotrophoblast zone to GC S1 and SI Appendix, Fig. S4). As no differences were detected area (P ≤ 0.05) (Fig. 4), a result consistent with that reported by based on the interaction of treatment and sex, results from male others for BPA (19, 21). and female placentas were considered together. BPA exposure lowered concentrations of D-fructose and the minor metabolites β 5-HT and Dopamine Immunohistochemistry. As a positive control docosahexaenoic acid (DHA), sophorose (2-O- -D-glucopyranosyl- and to establish optimal conditions for immunohistochemistry D-glucose), and glycolic acid relative to controls (SI Appendix,Fig. (IHC), we first confirmed that the antibody raised against 5-HT S5). In BPS vs. control placentas, fatty acids were most affected, could be used to localize this neurotransmitter within neurons of with significant decreases in stearic acid, palmitic acid, DHA, the fetal brain (SI Appendix, Fig. S6 A and B). When these same octadecenoic acid, and hexadecanoic (palmitic). D-ribose increased in concentration, while glycolic acid showed a decrease (as it did in conditions were applied to immunolocalize 5-HT in placental BPA-exposed placentas). sections, it became clear that the highest expression was within trophoblast GCs of the junctional zone, although there was also Targeted Metabolomics of Neurotransmitters in Fetal Placental some expression within the labyrinth region as well (Fig. 5 A–C). Tissue. Maternal exposure to BPA and BPS significantly in- The percentage of serotonin-positive GCs was reduced in pla- creased placental concentrations of dopamine (Fig. 3A). Conversely, centas that had been exposed to BPA and BPS relative to con- there was a dramatic decrease in placental serotonin (5-HT) fol- trols (P = 0.01) (Fig. 5D). Moreover, overall staining intensity in lowing BPA or BPS exposure (Fig. 3B). BPA and BPS increased the 5-HT–positive GCs also appeared to be depleted (Fig. 5A vs. Fig. ratios of 5-hydroxyindoleacetic acid (5-HIAA; primary 5-HT me- 5 B and C). As there was no difference in the area occupied by tabolite) to 5-HT ∼25- and 10-fold, respectively (Fig. 3C). There was GCs, we infer that there was less 5-HT in the GC compartment

4644 | www.pnas.org/cgi/doi/10.1073/pnas.1919563117 Mao et al. Downloaded by guest on September 25, 2021 APPLIED BIOLOGICAL SCIENCES

Fig. 2. qPCR analyses of placental genes shown to be differentially expressed (DE) based on RNAseq analyses. qPCR analysis of the imprinted gene, Ascl2,was also analyzed as its expression may be affected by BPA (18). In the current results, it is also down-regulated in e12.5 placenta of BPA- but not BPS-exposed mice relative to controls. N = 10 to 16 biological replicates tested per group. *P ≤ 0.05.

of BPA/BPS-exposed mice than in controls, a result consistent negative correlations across datasets (Fig. 7B). The percentage with whole-placental data shown in Fig. 3B. of GCs positive for 5-HT, but not the concentration of 5-HT We established conditions for dopamine IHC on fetal brain itself, correlated positively (r ≥ 0.8) with both placental fruc- sections similarly to those developed for 5-HT (SI Appendix, Fig. tose and placental E2 concentrations and most intriguingly, ex- S6 C and D). As with 5-HT, the primary cells in the placenta pression of Calm4, Sfrp4, Rimk1b, Epdr1, Ear2, Guca2a, Gdf10, staining positively for dopamine were the GCs (Fig. 6 A–C). In contrast Cxc14, Coch, and Mmp3 (i.e., 10 of the 11 genes down-regulated to 5-HT, however, BPA/BPS exposure increased the percentage of by exposure to BPA/BPS). The one exception, Gm9513, was not dopamine-positive GCs (P = 0.001) (Fig. 6D) and increased the positively correlated with the number of 5-HT–positive GCs intensity of dopamine staining in those cells. These results are when a cutoff of r ≥ 0.8 was used. E2 concentrations correlated consistent with the overall two- to threefold increase in placental strongly and positively with many of the same aspects of phenotype dopamine levels brought about by BPA/BPS exposure (Fig. 3C). as 5-HT–positive GCs. Examples include fructose concentration and expression of many of the same DEGs listed above. Correlations among Analytical Data. To examine associations be- By contrast, as the percentage of GCs positive for dopamine tween gene expression; changes in concentrations of metabolites, increased, there was a corresponding loss of 5-HT–positive GCs neurotransmitters, and steroid hormones; placental histology; and a negative correlation with expression of Calm4, Sfrp4, and 5-HT and dopamine immunoreactivity in GCs, we used the Rimk1b, Epdr1, Ear2, Guca2a, Gdf10, Cxc14, Coch, and Mmp3 mixOmics program (42) (SI Appendix, Fig. S7). This R-plot and fructose and E2 concentrations (Fig. 7B). Again, it was the analysis, which does not take into account directional changes, number of dopamine-positive cells rather than dopamine con- revealed a significant correlation (r = 0.61) between the observed centration per se that underpinned the correlations at r ≥ levels of polar metabolites and steroid hormone concentrations, 0.8. Finally, as the 5-HIAA:5-HT ratio increased, the area of suggesting a possible link between the shifts in metabolite levels spongiotrophoblast relative to GCs in the junctional zone brought about by BPA/BPS exposures and reduced estrogen shrank. concentrations. As inferred above, concentrations of the neurotransmitters, 5-HT and dopamine, correlated with the IHC data Discussion (r = 0.55 for 5-HT; r = 0.8 for dopamine). There was also a In humans, abnormal placental responses to EDCs, especially strong indication that the reduced area of spongiotrophoblast to BPA, have been proposed to lead to early pregnancy loss; pla- GCs noted in BPA/BPS-exposed placentas was linked to the cental diseases, such as preeclampsia and preterm birth; and changes in neurotransmitter levels (r = 0.52). adverse health effects on offspring after birth and into adult- We then generated circos plots by using the combined BPA/ hood, although none of these postulates have been definitively BPS data in order to provide greater power to the analyses (Fig. proven (43). However, it is becoming apparent in rodents that 7A), and this approach revealed more detailed positive and placental responses to in utero environmental insults can shape

Mao et al. PNAS | March 3, 2020 | vol. 117 | no. 9 | 4645 Downloaded by guest on September 25, 2021 Fig. 3. Effects of BPA and BPS on the placental production of neurotransmitters and E2 at e12.5. (A) Both BPA and BPS increased placental dopamine concentrations. *P = 0.01; **P = 0.03. (B) Both EDCs reduced placental production of serotonin (5-HT). *P = 0.0001; **P = 0.0006. (C) The placental 5-HIAA:5- HT ratio was increased with both chemicals. *P = 0.0001; **P = 0.001. (D) Placental E2 concentrations were reduced in BPA vs. control group. N = 10 to 16 biological replicates per group. *P = 0.01.

fetal brain development and postnatal behaviors of offspring statistical treatment of data. Perhaps most reassuring was that through the placental–brain axis (1, 36). Although the Food and BPA and BPS exposures both regulated a small set of 13 genes, Drug Administration has not banned BPA outright in response leaving the vast majority of other expressed genes unaffected. to concerns, especially in relation to human infant expo- This outcome strongly suggests that the chemicals targeted the sures, manufacturers have sought alternatives, one of which is placental transcriptome narrowly and selectively. the structurally related chemical, BPS (SI Appendix, Fig. S1). What particular roles any of the genes that are most influ- Here, we have sought to compare the effects of BPA and BPS enced by BPA and BPS might play in the development of fed to pregnant mice on the phenotype of the e12.5 placenta, a spongiotrophoblast and GCs (Fig. 4) are unclear. In our exper- stage when all of the essential features of the functionally mature iments, we detected a reduction in spongiotrophoblast to GC placenta are evident. Our general conclusion is that BPA and area with both treatments. BPA/BPS-induced differences in BPS exert remarkably similar effects on placental gene expression, these placental cellular components may at least partially ac- metabolome, and placental organization as well as on the content count for changes in gene expression in response to these of the neurotransmitters, dopamine and 5-HT, raising the likeli- treatments. Other studies agree that placentas from pregnant hood that the substitute chemical is potentially as harmful as its mice exposed to BPA show degenerative changes in spongio- predecessor. trophoblast and/or GC layers (19, 21). A similar reduction of Past studies in rodents, mainly in mice, suggest that BPA can spongiotrophoblast area was evident in mice with a partial loss affect expression of several genes in the placenta (13–16, 18, 20, of Ascl2 (also called Mash2), a maternally imprinted gene in 44, 45). Some of these reports focused on a few select candidates the IC2 cluster (40). Homozygous mutant mice that lack this (14–16, 18, 20, 44, 45), especially ones known to be imprinted gene exhibit a complete loss of spongiotrophoblast cells and −/+ (14, 18), while others used a global approach based on micro- die by e10 (48), while heterozygous Ascl2 mice show extra array analyses (13, 20) but were often underpowered. In addi- layers of parietal (external to the junctional zone) GCs (48, tion, doses of BPA fed to the mother varied as did the time in 49). Exposure to a very high dose of BPA (10 mg/kg body pregnancy when the placentas were analyzed. Whatever the weight per day) but not a lower dose (10 μg/kg body weight per reasons, none of these earlier studies on BPA placental effects day) has been reported to result in loss of imprinting and in- aligned with ours in terms of DEGs. One similarity with two creased expression of Ascl2 from what should have been the earlier studies, however, is that the WNT signaling pathway, repressed allele in e9.5 placenta (18). We failed to detect any which is important in the functional development of trophoblast changes in Ascl2 expression relative to controls by RNAseq analysis, in both the human (46) and the mouse (47), appeared affected by although there was a hint of decreased expression in the qPCR BPA and BPS (21, 22). We have confidence in our study because measurements (Fig. 2). It remains unclear, therefore, whether the it was robust both with regard to the number of samples analyzed reduction in spongiotrophoblast area associated with BPA and BPS (N = 10 to 16 biological replicates per treatment) and in the exposures involve Ascl2.

4646 | www.pnas.org/cgi/doi/10.1073/pnas.1919563117 Mao et al. Downloaded by guest on September 25, 2021 APPLIED BIOLOGICAL SCIENCES

Fig. 4. Histological images of placentas from control and BPA- and BPS-exposed mice. A–C depict the three main regions of the fetal placenta: labyrinth (LA), spongtiotrophoblast (spongioTB), and GCs. The areas of each these regions were determined as well as the ratio of the areas to each other. (D) BPA and BPS exposure reduces the spongioTB to GC ratio, suggestive that these trophoblast lineages are vulnerable to BPA/BPS exposure. N = 10 to 16 individuals (males and females combined) per group. *P ≤ 0.05.

One gene with expression that was down-regulated in both caused reductions in stearic and palmitic acids, which are also BPA- and BPS-exposed placentas was Gm9513, which belongs to important precursors of brain lipid components. Thus, exposure the Pate gene family, of which there are 14 members in the to either chemical could impact the placental–brain axis of the mouse and 4 in the human (50). Specifically, Gm9513 appears to fetus by indirectly reducing the concentrations of a few key correspond to Pate11 (originally Pate P) and is a possible metabolites. Importantly, however, the variance in the metab- ortholog of the human PATE 1 gene. Murine PATE11 seems to olome data may have precluded significant shifts in other me- be relatively specific to the rodent placenta (rather than prostate tabolites from being noted. and testis). PATE-like proteins have been implicated in having Perhaps the most dramatic effects of both BPA and BPS ex- neuroregulatory roles and appear localized to cells with a neu- posure were to reduce the concentration of 5-HT by over 80%, roendocrine phenotype (51). At this point, however, the possible raise that of its immediate metabolite 5-HIAA, and increase the Gm9513 roles of and those of the other 10 genes down-regulated concentration of a second neurotransmitter, dopamine, three- to Calm4 Sfrp4 Rimk1b Epdr1 Ear2 Guca2a by BPA/BPS ( , , , , , , fivefold (Fig. 3), while the third neurotransmitter measured, Gdf10 Cxc14 Coch Mmp3 , , ,and ) in regulating placental GABA, was not affected (SI Appendix,TableS6). 5-HT has been development are unclear. Notably, however, the reduction in – previously reported to be present in the mouse placenta from e9 spongiotrophoblast to GC area, the loss of 5-HT positive GCs, through e12 (58), with its presence largely limited to the GCs after and gain in dopamine-positive GCs do not seem to be strongly e10.5 (59). One crucial role for placental 5-HT is thought to be in correlated with the down-regulation of Gm9513 but instead, with the development of the fetal forebrain (60) by providing a vital the other 10 genes (Fig. 7). Of these, Sfrp4 (Secreted Frizzled supplementary source of the neurotransmitter. Hence, a reduction Protein 4) was most dramatically silenced by BPA/BPS exposure in the number of 5-HT–positive GCs and in total 5-HT placental (Table 1) and is a soluble inhibitor of WNT signaling, a pathway that participates in trophoblast lineage development (52). levels might starve the fetal brain at a critical time in its matura- −/− Sfrp4 mutant female mice produce fewer pups per litters than tion, leading to long-term defects in behavior that have previously controls (53), but the basis of this phenotype and whether it is been associated with BPA exposure. By contrast to 5-HT, there is associated with placentation are unknown. There is a similar lack little information relating to the occurrence of dopamine in the of information with regard to a role for the other genes in mouse placenta, although its presence in amniotic fluid has been placentation. inferred to have a placental origin (61). Our data demonstrate The metabolomics studies on polar metabolites in the placenta that dopamine is present in the mouse placenta in amounts indicated that BPA and BPS had minor effects, with only two comparable with those of 5-HT (Fig. 3) and that, like 5-HT, metabolites, DHA and glycolic acid, significantly down-regulated dopamine is highly concentrated in GCs within the junctional by exposure to both chemicals. DHA, in particular, is essential zone (Fig. 6). Whether there are classes of trophoblast GCs that for normal fetal brain development and regulation of levels of can be distinguished by their 5-HT vs. dopamine content re- neurotrophin and nerve growth factor (54–57). BPS but not BPA mains to be determined.

Mao et al. PNAS | March 3, 2020 | vol. 117 | no. 9 | 4647 Downloaded by guest on September 25, 2021 Fig. 5. Serotonin (5-HT) immunoreactivity in GCs. The 5-HT immunoreactivity in control (A), BPA (B), and BPS (C) GCs. (D) Determination of the percentage of 5-HT immunoreactivity GCs reveals that both BPA and BPS have decreased percentages of positively stained cells relative to control. Arrows indicate 5-HT positive immunoreactive GCs. N = 6 individuals per treatment group. *P = 0.01.

Two mouse models have been used to examine the role of 5-HT (Fig. 7), including, for example, the positive correlations observed in mouse placentation. One is the tryptophan hydroxylase (Tph1) between the majority of the DEGs, numbers of 5-HT–positive knockout mouse, which is unable to synthesize 5-HT (62), while GCs, and placental estradiol and fructose concentrations. From the second lacks the Solute Carrier Family 6 Member 4 these and other data discussed earlier, we infer, as others have (SLC6A4), a transporter required for uptake of 5-HT (62). Both done before (19, 21), that the target of BPA/BPS exposure is lo- strains of mice had slightly smaller placentas than their wild-type cated in the junctional zone of the rodent placenta, most likely counterparts with abnormalities mainly confined to spongio- either the trophoblast GCs or spongiotrophoblast adjacent to trophoblast of the junctional zone. In other words, these knock- them. As GCs are enriched for steroidogenic enzymes (65–68) and outs provide similar phenotypes to BPA/BPS exposures. Together, E2 may have a role in regulating expression of genes, such as these and other studies suggest that 5-HT is needed for tropho- tryptophan hydroxylase-2 (Tph2), Maoa, Maob, Sert,and5-HT blast maintenance (63), especially within the junctional zone, and receptor 1a (Htr1a) (69), declining E2 concentrations in GCs that the deleterious effects of BPA/BPS on spongiotrophoblast to could trigger reductions in 5-HT production and secondary GC area may be due, at least in part, to the lack of availability of changes in spongiotrophoblast that relies on GCs for neuroen- 5-HT from the adjacent GCs. Interestingly, although dopamine docrine support. One previous study in rats has shown that BPA itself has not been shown to accumulate in the rat placenta, the exposure reduces P450 aromatase activity in the ovary (70), but the predominant trophoblast cells that express dopamine D1 and D2 gene encoding this protein was not among those regulated in the receptors are spongiotrophoblast and GCs of the junctional zone present placental study. It should also be noted that BPA and BPS (64), raising the possibility that the locally increased concentra- effects on total placental E2 concentrations were modest and that tions of dopamine, in addition to the reduction in 5-HT, might only the data for BPA were significant. Importantly, however, the contribute to the observed pathology caused by BPA/BPS exposure in the mouse. levels of E2 (as well as those of fructose and all other metabolites) Whether the changes in concentrations of 5-HT or dopamine were derived from whole-placental extracts. Local effects in the caused as a result of BPA/BPS exposures are on the biosynthetic spongiotrophoblast and GCs might be considerably more dramatic, or degradative pathways is unclear at present, although the ac- thereby explaining many of the strong correlations seen in Fig. 7. cumulation of 5-HIAA could imply that 5-HT was being me- While maternal or fetal serum BPA/BPS concentrations in the tabolized at a higher rate in the exposed placentas. However, current studies were not assessed because of technical issues there were no significant alterations in the transcript levels of the arising from storing the frozen tissues in polypropylene tubes, the “ genes implicated in either the biosynthesis (e.g., tryptophan dose of BPA used was within the presumptive no observed hydroxylase [TPH1 and -2], dihydroxyphenylalanine [DOPA] adverse effect level” (NOAEL) (71–75) that yields serum con- decarboxylase [DDC], tyrosine hydroxylase [TH]) or degra- centrations comparable with those identified in human pop- dation (e.g., monoamine oxidases [MAO], catechol-O-methyl ulations (5, 10, 76–79). The BPS dose was chosen based on transferase [COMPT]) of 5-HT and dopamine. previous reports suggesting that it caused changes in maternal Our multiomics integrative approach and the construction of and offspring behavioral patterns (80, 81). This BPS dose falls circos plots revealed that surprisingly robust correlations could well below the toxicological NOAEL of 10 mg/kg per day (82) be made between diverse features of the BPA/BPS phenotype and can thus be considered to be low.

4648 | www.pnas.org/cgi/doi/10.1073/pnas.1919563117 Mao et al. Downloaded by guest on September 25, 2021 Fig. 6. Dopamine immunoreactivity in GCs. Dopamine immunoreactivity in control (A), BPA (B), and BPS (C) GCs. (D) Determination of the percentage of APPLIED BIOLOGICAL SCIENCES dopamine immunoreactivity GCs reveals that both BPA and BPS have increased percentages of positively stained cells relative to control. Arrows indicate dopamine positive immunoreactive GCs. N = 6 individuals per treatment group. *P = 0.001.

The present studies were solely designed to examine how BPA/ Our study reinforces the concept that BPA or BPS exposure BPS exposure affects the midgestational placenta (e12.5). Although alter the allocation of the placental cells within the junctional zone we cannot be sure, it seems likely that the effects noted (e.g., on and suggests that this may be achieved by altering the balance of 5- neurotransmitter levels) persist until parturition, and it does not HT and dopamine in GCs. These observations imply that there seem unreasonable to infer that these changes directly influence fetal are likely to be associated effects on the fetal brain via the pla- brain development, possibly leading to permanent neurobehavioral cental–brain axis and raise further concerns about possible neu- effects. We and others have shown that developmental exposure of rodevelopmental and other disorders arising in exposed offspring, fetuses in various rodent models to BPA/BPS can alter later neurobehavioral responses (76, 80, 81, 83–87). To tease apart potential at least in rodents. Caution is suggested, however, in relating these BPA/BPS-induced effects through the placental–brain axis vs. direct inferences to the human whose placental development differs effects on the fetal brain, it may be necessary to create conditional from that of the mouse and where there is no obvious homolog for knockout mice that lack BPA/BPS-responsive genes in the GCs and the junctional zone and its component spongiotrophoblast and spongiotrophoblast. Such mice are presently though unavailable. GCs (88, 89).

Fig. 7. Circos plot correlations for BPA/BPS-exposed vs. control placenta that includes DEGs, 5-HT IHC, dopamine IHC, neurotransmitter concentrations, histological proportions of the placenta, steroid hormones, and significant metabolite differences. Correlation value was set to 0.8. Red lines in the center indicate a positive correlation (A), whereas blue lines indicate a negative correlation (B). Lines on the outside of the circle indicate whether the value was greater in BPA/BPS (blue) or controls (orange). For this analysis to work, each category has to include more than one end point. Thus, number of GCs, labyrinth to GC ratio, and placenta testosterone concentrations were included even though they did not show treatment differences.

Mao et al. PNAS | March 3, 2020 | vol. 117 | no. 9 | 4649 Downloaded by guest on September 25, 2021 Experimental Procedures antibody (ab6336; Abcam) diluted 1:80 (vol/vol) at 4 °C overnight. For negative controls, adjacent tissue sections were incubated with 1% (wt/vol) BSA Sections on chemicals and reagents, fetal PCR sexing, placental RNA isolation, in PBS. The slides were washed twice in 0.025% Triton X-100 in PBS (5 min Illumina TruSeq RNA library preparation, sequencing and analyses, qPCR, and each), incubated in 3% (vol/vol) H O in PBS for 30 min, and incubated for 60 metabolomics, including analysis of neurotransmitters, steroid hormones, 2 2 min at room temperature (20 to 21 °C) with horseradish peroxidase (HRP)– and integrative correlation analyses, are described in SI Appendix, Supplemental polymer secondary antibody (PI-9400; Vector Laboratories) diluted to 1:400 Methods. (vol/vol). Staining was visualized by using the 3,3’-diaminobenzidine (DAB) peroxidase substrate kit (SK-4100; Vector Laboratories) and DAB Enhancer Animals and Treatments. All animal experiments were approved by the (S1961; Dako). Sections were counterstained with Mayer’s hematoxylin University of Missouri Animal Care and Use Committee (Protocol #8693) and and mounted with Permount mounting medium (SP15-500; Thermo conformed to the NIH Guide for the Care and Use of Laboratory Animals Fisher Scientific). (90). Five- to six-week-old C57BL6J female and male mice (Jackson Labs) For dopamine immunohistochemistry, a similar procedure was followed were placed in polypropylene cages and fed a phytoestrogen-free diet except for the following. The primary antibody was a rabbit polyclonal against (AIN93G; Envingo). Females were provided a week habituation period be- dopamine (ab8888; Abcam) diluted 1:100 (vol/vol). The secondary antibody was fore treatments were initiated. They were then randomly selected to be in HRP anti-rabbit immunoglobulin G (IgG) (PI-1000; Vector Laboratories) diluted one of three treatments groups: 1) provided a daily Nabisco Nilla wafer 1:400 (vol/vol). μ (Nabisco) containing 70% ethanol (8- to 24- L volume as adjusted for body Sections were photographed with a Leica DFC290 Color Digital Camera weight that was air dried beforehand onto the wafer); 2) provided a daily mounted on a Leica DM 5500B Compound Microscope (Leica Camera Inc.). Images μ wafer containing BPA (200 g/kg body weight reconstituted in ethanol with taken from same tissue sections were stitched together by using Leica Appli- a similar volume as vehicle control and air dried onto the wafer; and 3) cation Suite software (V4.12; Leica). The GC region was delineated with Adobe provided a daily wafer containing BPS (200 μg/kg body weight reconstituted Photoshop (CC 2018; Adobe). Percentage of serotonin 5-HT–immunoreactive in ethanol with a similar volume as vehicle control and air dried onto the and dopamine-immunoreactive GCs vs. total number of GCs was used for wafer). The BPA dose falls below the diet-administered maximum nontoxic statistical analyses and graphing purposes. dose for rodents (200 mg/kg body weight per day), is within the presumptive – NOAEL (71 75), and yields serum concentrations comparable with those Statistical Analyses. All dependent variables were analyzed for normality by identified in human populations unknowingly exposed to this chemical (5, using the Wilk–Shapiro test (Statistical Analysis Systems [SAS] v9.4). Fetal sex – 10, 76 79). The BPS dose was chosen based on previous reports suggesting ratio data were analyzed by using the generalized linear model of the SAS that it disrupted maternal and offspring behavioral patterns (80, 81). This version 9.4 (PROC GENMOD procedure). The data were distributed as a binomial BPS dose falls well below the toxicological NOAEL of 10 mg/kg per day (82) and transformed by means of a logit-link function. The χ2 test was used to test and can thus be considered a low dosage. Females were weighed weekly, deviation from a 1:1 ratio (i.e., a value of 0.5 for the fraction of male fetuses) as and volume of ethanol, BPA, or BPS pipetted on the wafer was accordingly well as for differences in sex ratio between groups. The antilog of the logit adjusted. To minimize background BPA/BPS contamination, animals were and the antilog of the differences between logit estimates produced the housed in polypropylene cages with aspen shavings rather than corn cob odds and odds ratio, respectively. The χ2 test was also used to test differ- and provided glass water bottles. BPA/BPS was stored in amber glass bottles ences in pregnancy rates between groups. Number of implantation sites with aluminum foil around them to minimize exposure to light. Both and fetuses and gestational body weight gain were analyzed by using the chemicals were reconstituted in polypropylene tubes. After 2 wk of receiving PROC MIXED procedure in SAS version 9.4. Differences between groups one of these treatments, females were paired with potential breeder males were determined by the Fisher least significant difference. Individual dam and checked the next morning for a vaginal plug. When a vaginal plug was was considered the experimental unit and used as error term to control for observed, this was considered e0.5 postcoitus. If no vaginal plug was ob- potential litter effects. served in the morning, the males were placed in separate cages and then Placental estradiol, estrone, corticosterone, testosterone, progesterone, repaired that evening. Females were continued on the respective treatments GABA, dopamine, 5-HT, and 5-HIAA concentrations were logarithmically until they were humanely euthanized at e12.5. At this time, each uterine transformed to approach a normal distribution for data analysis. Data for the horn was incised, and the uterine position of each conceptus was de- dependent variables of placental hormone and neurotransmitter concen- lineated. Fetal tissue was collected for PCR sexing. Half of the fetal pla- trations; ratios between labyrinth, junctional zone, and GC areas in placenta; cental tissue (as determined by the discoid morphology) where the blood vessel size within the labyrinth region data; and percentage of 5-HT underlying uterine tissue was dissected away was frozen in liquid nitrogen, immunoreactive-positive GCs relative to total number of GCs were ana- and the other half of the fetal placenta with underlying uterine tissue was lyzed by the PROC MIXED procedure of SAS v9.4. Sources of variation con- fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline solution sidered were treatment, sex, and treatment × sex interaction with an (PBS) for histological analyses. individual mouse serving as the experimental unit to determine treatment effects. Gene expression data determined by qPCR analyses were normal- Placental Histology. Before embedding, placenta tissue for histology was ized by using combined average delta cycle threshold (dCt) values of three marked with tattoo ink to aid in orientation. All attempts were made to housekeeping genes Rpl7, Porl2a, Ubc andanalyzedbythePROCGLMpro- −ΔΔCt obtain a cross-section through all fetal placental layers. In those sections where cedure of SAS. Graphs are based, however, on 2 values relative to the such layers were only partially included or missing, the tissue block was control values with group (males and females combined) mean value set to one deparaffinized and reoriented until such sections could be obtained. In those for graphing purposes. To examine treatment × sex interactions, the group placentas where adequate orientation was not achieved, they were replaced mean for control males or females was then determined based on the with same-sex placental samples from the same dam. Sections of placenta for mean value of these sexes relative to the group mean value of one. For all histology were fixed overnight in a 4% PFA solution at 4 °C. They were then data, a P value of ≤0.05 was considered significant. All data are presented subjected to three washes in 1× PBS before being placed in histology cassettes as means ± SEM. (Fisher Scientific) and stored in 70% ethanol until being processed. Histo- logical sections (4- to 5-μm thick) were cut at the Idexx Co. Histology Lab- ACKNOWLEDGMENTS. R.M.R. is supported by National Institute of Child oratory and stained with periodic acid-Schiff (PAS)-Hematoxylin. Histological Health and Human Development (NICHD) Grant HD094937. G.T. is supported slides were viewed, and images were photographed under a Leica DM 5500B by NICHD Grant RHD096083A. C.S.R. is supported by National Institute of upright microscope with a Leica DFC290 color digital camera at the Uni- Environmental Health Sciences (NIEHS) Grant 1R01ES025547. We acknowl- edge NIH Shared Instrumentation Grant 1S10OD018141 (to N.D.D.) for versity of Missouri Cytology Core Facility. Morphometric analyses on the hormone analyses. We thank the University of Missouri Office of Research obtained images were performed with ImageJ, NIH (https://imagej.nih.gov/ij/). and the University of Florida Office of Research for their financial support The labyrinth, spongiotrophoblast, and GC areas were measured within each to the University of Missouri Metabolomics Center and the University of placental section. Florida Interdisciplinary Center for Biotechnology Research Proteomics and Mass Spectrometry Core, respectively. We are grateful for assistance pro- 5-HT and Dopamine Immunohistochemistry and Morphometric Analyses. vided by Michelle J. Farrington, Jessica A. Kinkade, and the undergraduate research assistants who assisted in taking care of the mice and with the Paraffin-embedded sections (5 μm) of placental tissue were prepared on tissue collections. We are also grateful to the staff at the University of glass slides, deparaffinized in xylene, and rehydrated in a graded series of Missouri Cytology Core Facility who assisted with obtaining the placenta ethanol. Sections were subsequently incubated at room temperature (20 to histological images. We thank Dr. Kim-Anh Lê Cao and Abolfazl Jalal 21 °C) with blocking buffer containing 10% (vol/vol) normal serum with 1% Abadi, University of Melbourne, for their assistance with the mixOmics (wt/vol) bovine serum albumin (BSA) for 2 h and with primary anti–5-HT program.

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